engleski

Early Endosomal GTPase Rab5 Regulates GPIb Trafficking and Platelet Production In Vitro

Objective: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3- monophosphate (PI3P) production important for binding of effector proteins during early-to-late endosomal/lysosomal (LE/Lys) maturation. Approach and Results: To investigate the role of Rab5 in MKs, we expressed GFP-coupled Rab5 WT and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived MKs. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing MKs, when compared to GFP or Rab5 WT. Moreover, trafficking of GPIbß, a subunit of major MKs receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbß was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 WT MKs, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbß plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in LE/Lys. Localization of GPIbß in enlarged endosomes was PI3P dependent. Conclusion: Taken together, our results demonstrate that Rab5- dependent endocytosis plays an important role in MK receptor trafficking, membrane formation, and thrombopoiesis.

blood platelets ; endosomes ; thrombopoiesis

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